The mu opioid receptor, the type most closely associated with both analgesia and behavioral reinforcement, is subject to a variety of factors that regulate its effectiveness in eliciting a cellular response. Among these are the structure of the receptor itself, its density in the cell membrane, and its proximity to coupling proteins. In addition, these factors are all linked by dynamic interactions whose timing, only partially understood at present, can have important influence on the performance of a drug. During the past year we have continued investigations into these factors and dynamics in two ways. The first employed confocal microscopy and a high affinity fluorescent antagonist, "FNAL". The second is the ongoing study of the irreversible antagonist beta-FNA to determine whether a mutant receptor, H297Q, remains capable of covalent modification by the drug and how its response to the drug differs from that of the wild-type receptor. The results of the two programs follow: (1) Confocal microscopy is not only useful to localize probes at a subcellular level, but it can also quantitate them on living cells in real time, as we first showed in 2000. The method is unique in its ability to optically section biological material, and we employ this capability to demonstrate binding kinetics that depend on the vertical position of a cell's membrane. This year we have perfected the technique in a number of ways including the implementation of an "autofocus" macro that permits long term experiments with near perfect z-plane stability. In addition we now employ fast solution flow to eliminate kinetic confounds of binding with solution exchange. We were joined by Richard Shrager, whose expertise in kinetic modeling is guiding the experiments and disentangling the results. (2) The kinetic studies with beta-FNA have continued this year. We have previously seen that kinetic rate constants change with the mutant receptor (H297Q), but we are now pursuing whether that change is a consequence of the mutation or the lower expression level of that phenotype. In addition to these experiments, the project raised the question of the effect of sodium on receptor signal transduction. We showed that when sodium is exchanged for equal concentrations of choline, maximum responses decrease and the half maximum concentrations show some tendency to shift leftward. Using flame photometry, we demonstrated that the incubation medium (sodium or choline) has no effect on the intracellular concentrations of sodium and potassium in Xenopus oocytes, the model cells used in these experiments, indicating that the effects of sodium are solely extracellular.